Lariat-Peptide MccJ25
The antibacterial peptide microcin J25 (MccJ25) inhibits bacterial RNA polymerase by binding within, and obstructing, the secondary channel of bacterial RNA polymerase (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). MccJ25 has the sequence Gly1-Gly2-Ala3-Gly4-His5-Pro6-Val7-Glu8-Tyr9-Phe10-Val11-Gly12-Ile13-Gly14-Thr15-Pro16-Ile17-Ser18-Phe19-Tyr20-Gly21 cyclic(8→1) peptide (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 1, line 1).
MccJ25 has an unusual “lariat-peptide” covalent structure (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 1, line 1). MccJ25 is 21 amino acids in length and consists of an 8-residue cyclic segment—with a backbone-sidechain amide linkage between the backbone nitrogen atom of residue Gly1 and the side-chain carboxyl group of Glu8—followed by a 13-residue linear segment.
MccJ25 further has an unusual “lariat-protoknot” three-dimensional structure (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483; FIG. 2). In the three-dimensional structure of MccJ25, the linear segment of the lariat loops back and penetrates and threads through the cycle of the lariat, essentially as a thread through the eye of a needle. The linear segment is irreversibly locked in place and trapped within the cycle by steric constraints imposed by the aromatic sidechains of Phe19 and Tyr20, which bracket the cycle, with the aromatic sidechain of Phe19 being on located on one face of the cycle and the aromatic sidechain of Tyr20 being located on the other face of the cycle.
The lariat-peptide/lariat-protoknot structure of MccJ25, with irreversible trapping of the linear segment of the lariat within the cycle of the lariat, results in exceptional resistance to denaturation (complete resistance to tested thermal and chemical denaturants; Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483) and exceptional resistance to proteolysis (complete resistance to tested mesophilic endo- and exopeptidease; Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483).
The lariat-peptide/lariat-protoknot structure of MccJ25 is generated by a MccJ25-specific biosynthetic system. MccJ25 is produced by bacterial strains harboring a plasmid-borne lariat-peptide/lariat-protoknot biosynthetic cassette, consisting of a gene for MccJ25 precursor, two genes for factors that process McJ25 precursor into mature MccJ25, and one gene for a factor that exports MccJ25 from the cell (Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662). The gene names and functions are as follows:
mcjA; encodes the MccJ25 precursor (McjA)
mcjB; encodes a MccJ25 processing factor (McjB)
mcjC; encodes a MccJ25 processing factor (McjC)
mcjD; encodes a MccJ25 export factor (McjD)
The MccJ25 precursor is a 58-residue peptide consisting of a 37-residue N-terminal pro-sequence (residues numbered as −37 to −1) and a 21-residue C-terminal segment having the same amino acid sequence as mature MccJ25 (residues numbered as 1 to 21) (Solbiati, et al. (1999) J. Bacteriol 181, 2659-2662).
Processing of the MccJ25 precursor to yield mature MccJ25 entails two reactions: (1) cleavage of the backbone-backbone amide linkage between residue −1 and residue 1, resulting in removal of the 37-residue N-terminal pro-sequence; and (2) formation of a backbone-sidechain amide linkage between the backbone nitrogen atom of residue 1 and the sidechain carboxyl of residue 8, resulting in cyclization of residues 1-8 and entrapment of residues 9-21 (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). The MccJ25 processing factor McjC exhibits amino acid sequence similarity to amidotransferases of the Asn-synthase/Gln-hydrolase class, which catalyze transfer of ammonia or an amine from an amide donor to a carboxyl acceptor (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383). It has been proposed that McjC participates in both reactions in processing of the MccJ25 precursor to yield mature MccJ25, acting on pre-folded MccJ25 precursor to catalyze transfer of the backbone nitrogen atom, also known as the α-amino group, of residue 1 from the backbone amide linkage between residue −1 and residue 1 to the sidechain carboxyl of residue 8 (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383).
It has not been possible to date to re-create the lariat-peptide/lariat-protoknot structure of MccJ25 without use of the above-described MccJ25-specific biosynthetic system. Attempted chemical synthesis of MccJ25 yields a product having a lariat-peptide covalent structure but not having a lariat-protoknot three-dimensional structure (i.e., not having the linear segment of the lariat looped back and penetrating and threading through the cyclic segment of the lariat; the resulting material exhibits no detectable biological activity (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). Chemical synthesis of a linear analog of MccJ25 yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure; the resulting material exhibits no detectable biological activity (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483). Production of a recombinant linear analog of MccJ25, by use of a nucleic acid sequence encoding the linear analog of MccJ25 likewise yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure and exhibiting no detectable biological activity.
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette is organized as a gene cluster, with gene order mcjA, mcjB, mcjC, mcjD (Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662). The size of the MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette is ˜4,500 bp.
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette can be expressed in an original, naturally occurring, Mcc25-producer strain, resulting in MccJ25 production (Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662).
The MccJ25 lariat-peptide/lariat-protoknot biosynthetic cassette also can be introduced into, and expressed in, a surrogate host strain, resulting in surrogate-host MccJ25 production (Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662; Blond, et al. (1999) Eur. J. Biochem. 259, 747-755).
Because the nucleotide sequence of the mcjA gene determines the amino acid sequence of the MccJ25 precursor, and because, in MccJ25 biosynthesis, the MccJ25 precursor is processed to yield mature MccJ25, a change in the nucleotide sequence of the mcjA gene (“mutation”) can result in a corresponding change in the amino acid sequence of MccJ25 (“substitution”). The relationship between a mutation in the mcjA gene and a corresponding substitution in MccJ25 is determined by, and is predictable from, the universal genetic code.
Introduction of a mutation into the mcjA gene can be accomplished in straightforward fashion by use of molecular-biology and directed-evolution procedures known in the art, including, for example, random mutagenesis, site-directed mutagenesis, and gene synthesis (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751; see also Sambrook, J., et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
Accordingly, mcjA derivatives containing mutations, and corresponding MccJ25 derivatives containing substitutions, can be, and have been, prepared (See WO 2004/023093 and U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006, the entire contents of each of which are incorporated by reference; and Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751). This approach is limited to production of MccJ25 derivatives containing substitutions at a subset of residue positions, since other residue positions cannot be substituted without disruption of processing or export (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). Nevertheless, this approach provides a source of MccJ25 derivatives having different useful properties, including, for example, high affinity for a target of interest, high potency for inhibition of a reaction of interest, or suitability for site-specific incorporation of a detectable group such as a fluorochrome (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751).
In the same manner, “libraries” of mcjA derivatives containing single mutations or small numbers of mutations, and corresponding “libraries” of MccJ25 derivatives containing single substitutions or small numbers of substitutions, can be, and have been, prepared (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). This approach is limited to production of libraries of MccJ25 derivatives containing substitutions at a subset of residue positions, since other residue positions cannot be substituted without disruption of processing or export (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006). Nevertheless, this approach, optionally combined with screening of “libraries” by use of procedures known in the art, provides a further source of MccJ25 derivatives with different useful properties, including, for example, high potency for inhibition of a reaction of interest, or suitability for site-specific incorporation of a detectable group such as a fluorochrome (U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006).
MccJ25 inhibits Gram-negative bacterial RNA polymerase by binding within, and obstructing, the secondary channel of Gram-negative bacterial RNA polymerase (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). Through inhibition of Gram-negative bacterial RNA polymerase, MccJ25 exhibits antibacterial activity against certain Gram-negative bacterial species, including the Gram-negative enterics Escherchia coli and Salmonella sp. (Delgado, et al. (2001) 183, 4543-4550; Yuzenkova, et al. (2002) 277, 50867-50875). MccJ25 does not inhibit Gram-positive bacterial RNA polymerase or Thermococcus-Deinococcus bacterial RNA polymerase, and, accordingly, MccJ25 not inhibit Gram-positive bacterial RNA polymerase or Thermococcus-Deinococcus bacterial growth (Yuzenkova, et al. (2002) 277, 50867-50875).
The binding site for MccJ25 within bacterial RNA polymerase is remote from the binding site for rifamycins and from the sites of substitutions that confer resistance to rifamycins (WO 2004/023093; Mukhopadhyay et al. (2004) Mol. Cell 14, 739-751; Adelman, et al. (2004) Mol. Cell 14, 753-762). Accordingly, MccJ25 exhibits no cross-resistance with rifamycins and retains full ability to inhibit RNA polymerase derivatives resistant to rifamycins.
MccJ25, as a direct consequence of its lariat-peptide/lariat-protoknot structure, exhibits two features useful for drug design and drug discovery:
(1) MccJ25 is genetically encoded (albeit indirectly, through genetic encoding of a precursor and processing and export factors; Solbiati, et al. (1996) J. Bacteriol. 178, 3661-3663; Solbiati, et al. (1999) J. Bacteriol. 181, 2659-2662), permitting efficient production by fermentation (Blond, et al. (1999) Eur. J. Biochem. 259, 747-755) and permitting efficient construction of derivatives by molecular-biology or directed-evolution procedures (WO 2004/023093; U.S. application Ser. No. 11/371,736, filed Mar. 9, 2006; Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751).(2) MccJ25 is resistant to denaturation and proteolysis (Bayro, et al. (2003) J. Am. Chem. Soc. 125, 12382-12383; Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483).
This combination of features is not only useful, but also unusual. Most compounds do not exhibit this combination of features. (Most peptides and proteins exhibit only the first feature. Most non-peptide, non-protein compounds exhibit only the second feature.)
Non-MccJ25-Related Lariat Peptides
We refer herein to compounds according to general structural formula (I) as “non-MccJ25-related lariat peptides.”
wherein:                (i) X is an amino acid residue containing a backbone nitrogen atom;        (ii) Y is an amino acid residue containing a side-chain carboxyl group;        (iii) α is a peptide segment of from about 5 to about 8 amino acid residues;        (iv) β is a peptide segment of from about 6 to about 15 amino acid residues;        (v) there is an amide bond between the backbone nitrogen atom of X and the side-chain carboxyl of Y; and        (vi) wherein the compound has less than 25% amino acid sequence identity with microcin J25 (MccJ25).        
Non-MccJ25-related lariat peptides are known in the art and include the siamycins [siamycin I (also known as MS-271, NP-06, and FR901724; Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517), siamycin II (Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286), and siamycin III (also known as RP 71955 and aborycin; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743)], RES-701-n [RES-701-1 (Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280), RES-701-2 (Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220), RES-701-3 (Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220), and RES-701-4 (Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220)], propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), anantin (Wyss, et al. (1991) J. Antibiot. 44, 172-180), and lariatins [lariatin A and lariatin B (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491)] (FIG. 1).
Known non-MccJ25-related lariat peptides have lariat-peptide covalent structures similar to the lariat-peptide covalent structure of MccJ25 (Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743; Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220; Kimura, et al. (1997) J. Antibiot. 50, 373-378; Wyss, et al. (1991) J. Antibiot. 44, 172-1801; Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491; FIG. 1). The known non-MccJ25-related lariat peptides have lengths of 16-21 residues (FIG. 1). The known non-MccJ25-related lariat peptides contain either: (1) an 8-residue cyclic segment—with a backbone-sidechain amide bond between the backbone nitrogen atom of Xaa1 and the sidechain carboxyl of Glu8 or Asp8-followed by a 9- to 12-residue linear segment; or (2) a 9-residue cyclic segment—with a backbone-sidechain amide bond between the backbone nitrogen atom of Xaa) and the sidechain carboxyl of Asp9-followed by a 7- to 12-residue linear segment (FIG. 1).
Known non-Mcd25-related lariat peptides have lariat-protoknot three-dimensional structures similar to the lariat-protoknot three-dimensional structure of MccJ25. Three-dimensional structures have been determined for several known non-MccJ25-related lariat peptides, including siamycin I (Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129), siamycin II (Constantine, et al. (1995) J. Biomol. NMR 5, 271-286), siamycin III (Frechet, et al. (1994) Biochem. 33, 42-50)], RES-701-1 (Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280), and lariatin A (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491). In each case, the compound has been found to have a lariat-protoknot structure, in which the linear segment of the lariat loops back, and penetrates and threads though the cyclic segment of the lariat (Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; (Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Frechet, et al. (1994) Biochem. 33, 42-50); Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491).
The known non-MccJ25-related lariat peptides have been reported to have useful properties, including antibacterial activity for siamycins (Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743), propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), and lariatins (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491); antiviral activity for siamycins (Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Lin, et al. (1996) Antimicrob. Agents Chemother. 40, 133-138); bacterial RNA polymerase inhibition activity for siamycins (PCT Application Serial No., filed Mar. 13, 2007), RES-701-n (PCT Application Serial No., filed Mar. 13, 2007), and propeptin (PCT Application Serial No., filed Mar. 13, 2007); endothelin type B receptor antagonist activity for RES-701-n (Morishita, et al. (1994) J. Antibiot. 47, 269-275; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220); prolyl endopeptidase inhibition activity for propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378); and atrial natriuretic factor receptor antagonist activity for anantin (Wyss, et al. (1991) J. Antibiot. 44, 172-1801).
It is disclosed in PCT Application Serial No. PCT/US2007/006282, filed Mar. 13, 2007 that known non-MccJ25-related lariat peptides, including siamycins, RES-701-n, and propeptin, inhibit bacterial RNAP.
It further is disclosed in PCT Application Serial No. PCT/US2007/006282 that known non-MccJ25-related lariat peptides, including siamycins, RES-701-n, and propeptin, inhibit Gram-negative bacterial RNAP.
It further is disclosed in PCT Application Serial No., filed Mar. 13, 2007 that known non-MccJ25-related lariat peptide, including siamycins, RES-701-n, and propeptin, inhibit Gram-positive bacterial RNAP. This is in contrast to MccJ25, which does not inhibit Gram-positive bacterial RNAP (Yuzenkova, et al. (2002) 277, 50867-50875).
It further is disclosed in PCT Application Serial No. PCT/US2007/006282 that known non-MccJ25-related lariat peptide, including siamycins, RES-701-n, and propeptin, inhibit Thermus-Deinoccoccus bacterial RNAP, including, for example, Thermus thermophilus RNAP. This is in contrast to MccJ25, which does not inhibit Thermus-Deinoccoccus bacterial RNAP (Yuzenkova, et al. (2002) 277, 50867-50875).
It further is disclosed in PCT Application Serial No. PCT/US2007/006282 that known non-MccJ25-related lariat peptide, including siamycins, RES-701-n, and propeptin, are useful as broad-spectrum inhibitors of bacterial RNAP, being able to inhibit all three classes of bacterial RNAP: i.e., Gram-negative bacterial RNAP, Gram-positive bacterial RNAP, and Thermus-Deinoccoccus bacterial RNAP. This is in contrast to MccJ25, which is a narrow-spectrum inhibitor of bacterial RNAP, being able to inhibit only Gram-negative bacterial RNAP (Yuzenkova, et al. (2002) 277, 50867-50875).
The known non-MccJ25-related lariat peptides exhibit no significant amino acid similarity to MccJ25 (less than 25% sequence identity; FIG. 1).
The known non-MccJ25-related lariat peptides are produced by bacterial producer strains: Streptomyces sp. strains for siamycins, RES-701-n, and anantin (Yano, et al. (1996) Bioorg. Med. Chem 4, 115-120; Katahira, et al. (1996) Bioorg. Med. Chem 4, 121-129; Chokekijchai, et al. (1995) Antimicrob. Agents Chemother. 39, 2345-2347; Nakashima, et al. (1996) Biol. Pharm. Bull. 19, 405-412; Detlefsen, et al. (1995) J. Antibiot. 48, 1515-1517; Constantine, et al. (1995) J. Biomol. NMR 5, 271-286; Helynck, et al. (1993) J. Antibiot. 46, 1756-1757; Frechet, et al. (1994) Biochem. 33, 42-50; Potterat, et al. (1994) Liebigs Annalen der Chemie 7, 741-743; Yamasaki, et al. (1994) J. Antibiot. 47, 276-280; Katahira, et al. (1995) Bioorg. Med. Chem. 3, 1273-1280; Yano, K. et al. (1995) J. Antibiot. 48, 1368-1370; Ogawa, et al. (1995) J. Antibiot. 48, 1213-1220; Wyss, et al. (1991) J. Antibiot. 44, 172-1801), a Microbispora sp. strain for propeptin (Kimura, et al. (1997) J. Antibiot. 50, 373-378), and a Rhodococcus sp. strain for lariatins (Iwatsuki, et al. (2006) J. Am. Chem. Soc. 128, 7486-7491).
Low yields of non-MccJ25-related lariat peptides upon fermentation of the Streptomyces sp., Microbispora sp., and Rhodococcus sp. producer strains have complicated preparation and use of non-MccJ25-related lariat peptides.
It has not been possible to date to re-create the lariat-peptide/lariat-protoknot structure of a non-MccJ25-related lariat peptide without use of the Streptomyces sp., Microbispora sp., and Rhodococcus sp. producer strains. Attempted chemical synthesis of RES-701-1 yields a product having a correct lariat-peptide covalent structure but not having a correct lariat-protoknot three-dimensional structure (i.e., not having the linear segment of the lariat looped back and penetrating and threading through the cyclic segment of the lariat); the resulting material exhibits only 1/700 the biological activity of authentic RES-701-1 (Katahira, et al. (1995) Bioorg. Med. Chem. Lett. 5, 1595-1600; He, et al. (1995) Bioorg. Med. Chem. Lett. 5, 621-626). Chemical synthesis of a linear analog of RES-701-1 yields a product having neither a lariat-peptide covalent structure nor a lariat-protoknot structure; the resulting material exhibits no detectable biological activity (He, et al. (1995) Bioorg. Med. Chem. Lett. 5, 621-626). These negative results are reminiscent of negative results obtained for attempted chemical synthesis of MccJ25 and for chemical synthesis and recombinant production of a linear analog of MccJ25 (Rosengren, et al. (2003) J. Am. Chem. Soc. 125, 12464-12474; Wilson, et al. (2003) J. Am. Chem. Soc. 125, 12475-12483)
No lariat-peptide/lariat-protoknot biosynthetic cassettes for non-MccJ25-related lariat peptides have been described in prior art. As a consequence, surrogate-host expression of non-MccJ25-related lariat peptides has not been accomplished in prior art. As a further consequence, production of substituted derivatives of non-MccJ25-related lariat peptides has not been accomplished in the prior art. As a further consequence, production of libraries of substituted derivatives of non-MccJ25-related lariat peptides has not been accomplished in prior art. Accordingly, there is exists a need to address these shortcomings in the prior art.